PROJECT SUMMARY (Abstract) The Nuclear Pore Complex (NPC) is a large cylindrical assembly embedded in the nuclear envelope and comprised of proteins termed Nups. It is central for nuclear function at two related levels. First, as a regulator of transport, the NPC controls signaling access to the DNA and the passage of genetic information from DNA. Second, the NPC is an important regulator of genes by binding chromatin and its regulators to control expression states, a phenomenon that is poorly understood at the molecular level. These pivotal roles in all eukaryotic cells involve dozens of interacting pathways influencing virtually all aspects of cellular function. As a consequence, disruption of the NPC leads to many human disorders. Despite this, and though the nuclear transport machinery is a valid and powerful drug target, the NPC and the nuclear transport machinery have not been a significant part of therapeutic strategies. Arguably, there are two fundamental reasons why this is the case: (i) we do not know enough about the structure of the NPC to predict its behavior; (ii) the nuclear transport machinery impacts a bewildering array of cellular functions - thus even with a deep understanding of its structure, we still require complementary functional information to be able to predict the outcome of the targeted disruption of key elements of the transport pathway. We propose two Specific Aims that inform each other in a synergistic fashion. First, we will perform structural mapping of disease-associated Nup complexes, focusing on components of the cytoplasmic export platform and inner rings that have been linked to oncogenic and developmental defects. We will use enhanced versions of the methods we have already successfully deployed to generate high-resolution maps of these two regions and their attachment sites. On completion of this study, we will have mapped most of the NPC at high precision, allowing the two regions to be seen in the context of the whole NPC assembly. Second, and in parallel, we will map the functions of disease-associated Nup complexes. We will dissect the functionalities associated with the target Nup complexes, and determine the defects associated with their alteration - testing the hypothesis that these Nups are linked to diseases because their disruption alters critical gene expression patterns in a manner distinct from other nucleoporins. Realizing these aims will generate NPC structure-function maps in unprecedented detail and which are essential to understanding how different parts of the NPC act together to determine its functionality. This project will shed light on the nature of numerous disorders associated with human NPC dysfunction; aimed ultimately to open the nuclear transport machinery to rational and predictive drug design.